Abstract
BackgroundAlthough C/EBPβko mice are refractory to Bleomycin-induced lung fibrosis the molecular mechanisms remain unknown. Here we show that blocking the ribosomal S-6 kinase (RSK) phosphorylation of the CCAAT/Enhancer Binding Protein (C/EBP)-β on Thr217 (a RSK phosphoacceptor) with either a single point mutation (Ala217), dominant negative transgene or a blocking peptide containing the mutated phosphoacceptor ameliorates the progression of lung injury and fibrosis induced by Bleomycin in mice.Methodology/Principal FindingsMice expressing the non-phosphorylatable C/EBPβ-Ala217 transgene had a marked reduction in lung injury on day-13 after Bleomycin exposure, compared to C/EBPβwt mice, judging by the decrease of CD68+ activated monocytes/macrophages, bone marrow-derived CD45+ cells and lung cytokines as well as by the normal surfactant protein-C expression by lung pneumocytes. On day-21 after Bleomycin treatment, C/EBPβwt mice but not mice expressing the dominant negative C/EBPβ-Ala217 transgene developed severe lung fibrosis as determined by quantitative collagen assays. All mice were of identical genetic background and back-crossed to the parental wild-type inbreed FVB mice for at least ten generations. Treatment of C/EBPβwt mice with a cell permeant, C/EBPβ peptide that inhibits phosphorylation of C/EBPβ on Thr217 (40 µg instilled intracheally on day-2 and day-6 after the single Bleomycin dose) also blocked the progression of lung injury and fibrosis induced by Bleomycin. Phosphorylation of human C/EBPβ on Thr266 (human homologue phosphoacceptor) was induced in collagen-activated human lung fibroblasts in culture as well as in activated lung fibroblasts in situ in lungs of patients with severe lung fibrosis but not in control lungs, suggesting that this signaling pathway may be also relevant in human lung injury and fibrosis.Conclusions/SignificanceThese data suggest that the RSK-C/EBPβ phosphorylation pathway may contribute to the development of lung injury and fibrosis.
Highlights
The morbidity and mortality, as well as the financial and personal burden of lung injury and fibrosis are substantial
We show that inhibition of C/EBPb phosphorylation on Thr217 (RSK phosphoacceptor) within the transactivation domain with either a single point mutation (Ala217), dominant negative transgene or a blocking peptide containing the mutated phosphoacceptor ameliorates the progression of lung injury and fibrosis induced by Bleomycin in mice
Given the important role of ribosomal S-6 kinase (RSK) and its phosphorylation of C/ EBPb-Thr217 in the activation of liver myofibroblasts [10],[22] and the suggested similarities between liver and lung fibrogenesis [1],[23], we hypothesized that a dominant negative, non-phosphorylatable transgenic C/EBPb-Ala217 would block phosphorylation of C/EBPb-Thr217 by RSK, preventing lung myofibroblast (LMF) activation and decreasing lung fibrosis after lung injury
Summary
The morbidity and mortality, as well as the financial and personal burden of lung injury and fibrosis are substantial. Lung fibrosis and the resulting abnormal pulmonary function are responsible for the associated complications, including pulmonary hypertension, cardiac failure, lung tumors, and cachexia [1]. All these complications of lung fibrosis are the main indication for lung transplantations in the US and worldwide [2]. We show that blocking the ribosomal S-6 kinase (RSK) phosphorylation of the CCAAT/Enhancer Binding Protein (C/EBP)-b on Thr217 (a RSK phosphoacceptor) with either a single point mutation (Ala217), dominant negative transgene or a blocking peptide containing the mutated phosphoacceptor ameliorates the progression of lung injury and fibrosis induced by Bleomycin in mice
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